TWI442920B - Usage of toona sinensis extract for preparing drugs for decreasing weight or body fat - Google Patents

Description

Use of camphor extract for drugs that reduce body weight or reduce body fat

The present invention relates to a pharmaceutical composition or a camphor extract for treating metabolic syndrome, and more particularly to a pharmaceutical composition or a camphor extract for reducing body weight or reducing body fat.

As the economy flourishes, human eating habits tend to become more refined, leading to modern people taking too much sugar and fat to cause obesity. The problem of obesity often leads to a variety of diseases, such as cardiovascular disease, type 2 diabetes, hepatobiliary disease, cancer and the like. Here, the so-called obesity mainly refers to the accumulation of excess fat in the body, and excessive fat affects the body's normal metabolic mechanism.

According to statistical studies, obese people in the lower abdomen are at high risk of cardiovascular disease, and obesity is also one of the factors that cause hypertension and diabetes. Therefore, obesity is indeed a high risk factor for cardiovascular disease. In addition, according to research by the International Agency for Research on Cancer, some of the factors that cause post-menopausal breast cancer, colorectal cancer, kidney cancer, esophageal cancer, and endometrial cancer are strongly related to overweight and obesity.

In addition to causing cardiovascular diseases and cancers mentioned above, obesity is one of the factors that induce fatty liver production, and especially those with abdominal obesity are most susceptible to fatty liver. The reason is that the abdomen The fat cells are susceptible to stimulation and release fatty acids into the blood and are delivered to the liver through the circulatory system. The liver system converts fatty acids into triglycerides, and the newly synthesized triglycerides bind to very low density lipoproteins in liver cells and are transported to the blood. When the rate of synthesis is greater than the rate of transport, or when it is unable to bind to very low density lipoproteins, it increases the risk of fatty liver.

Because obesity causes quite a lot of diseases, obesity has been listed as the primary concern of the 21st century by the World Health Organization. Accordingly, if you can lose weight or reduce body fat, it is bound to reduce the risk of suffering from the above diseases. Therefore, it is highly desirable to develop a pharmaceutical composition that can reduce body weight or reduce body fat for the purpose of treating obesity.

The main object of the present invention is to provide a pharmaceutical composition or a camphor extract for reducing body fat, which can reduce body fat synthesis in the body.

Another object of the present invention is to provide a pharmaceutical composition or a camphor extract for reducing body weight, which can reduce body weight and reduce the incidence of metabolic syndrome.

Another object of the present invention is to provide a method for preparing a citron extract which reduces body fat or reduces body weight, and which can extract an active ingredient which can reduce body fat or reduce body weight in a citron by a simple process.

In order to achieve the above object, the present invention provides a pharmaceutical composition for reducing body fat or reducing body weight, comprising: 40 to 75 parts by weight of gallic acid; 15 to 40 parts by weight of ethyl gallate; 10 to 30 parts by weight of methyl gallate.

In addition, the present invention also provides a camphor extract for reducing body fat or reducing body weight, which is obtained by the following steps: (A) providing a camphor leaf, and extracting the camphor leaf by using water to obtain a camphor extract. And filtering the first extract of the camphor to obtain a first solid phase extract; (B) extracting the first solid phase extract with an aqueous alcohol solution to obtain a second solid phase extract; (C) extracting the second solid phase extract with an aqueous alcohol solution to obtain a third solid phase extract; Extracting the third solid phase extract with an aqueous alcohol solution to obtain a fourth solid phase extract; (E) extracting the fourth solid phase extract with an aqueous alcohol solution to obtain a fifth solid phase extract; and (F) The fifth solid phase extract was extracted using an aqueous alcohol solution to obtain a citron extract. The extract of the camphor extract extracted through the above process is analyzed to have a component of the pharmaceutical composition as described above.

Accordingly, the pharmaceutical composition for reducing body fat or reducing body weight of the present invention comprises a citron extract comprising 40 to 75 parts by weight of gallic acid; 15 to 40 parts by weight. Ethyl gallate; and 10 to 30 parts by weight of methyl gallate.

In the pharmaceutical composition provided by the present invention, the composition of the composition is relatively simple, and the main components thereof are gallic acid and its esters. In the present invention, the effect of reducing body weight or reducing body fat can be attained by using a simple pharmaceutical composition of this composition. In addition, the citron extract provided by the present invention can extract the components such as gallic acid and its ester in the citron extract through a simple alcohol water extraction process. By using the extract of the camphor extract extracted by the present invention, the effect of reducing body weight or reducing body fat can be achieved. Since the toon extract of the present invention is an extract of a natural product, in addition to being used as a medical product, it can also be used as a daily health food to achieve the effect of controlling body weight or body fat.

In the pharmaceutical composition of the present invention, preferably, the content of gallic acid is 45 to 65 parts by weight, the content of ethyl gallate is 20 to 30 parts by weight, and the content of methyl gallate is It is 15 to 25 parts by weight. More preferably, the content of gallic acid is 50 to 60 parts by weight, the content of ethyl gallic acid is 20 to 30 parts by weight, and the content of methyl gallate is 15 to 25 parts by weight.

Further, the pharmaceutical composition of the present invention may further comprise: 1 to 10 parts by weight of musk Prime.

Furthermore, the method for preparing the tortoise extract for reducing body fat or reducing body weight provided by the invention comprises the following steps: (A) providing a camphor leaf, and extracting the camphor leaf by using water to obtain a camphor extract. And filtering the first extract of the camphor to obtain a first solid phase extract; (B) extracting the first solid phase extract with an aqueous alcohol solution to obtain a second solid phase extract; (C) extracting the second solid using an aqueous alcohol solution a solid phase extract to obtain a third solid phase extract; (D) extracting a third solid phase extract using an aqueous alcohol solution to obtain a fourth solid phase extract; (E) extracting a fourth solid phase using an aqueous alcohol solution Extracting to obtain a fifth solid phase extract; and (F) extracting the fifth solid phase extract using an aqueous alcohol solution to obtain a citron extract.

In the citron extract of the present invention and the preparation method thereof, the aqueous alcohol solution used in the step (B), the step (C), the step (D), the step (E), and the step (F) may be independently selected from methanol. An aqueous solution of the group consisting of ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, and mixtures thereof. Preferably, the aqueous alcohol solution used in the step (B), the step (C), the step (D), the step (E), and the step (F) are each independently an aqueous ethanol solution.

Further, in the camphor extract of the present invention and the method for producing the same, extraction is carried out using aqueous solutions of different concentrations of alcohol; and it is preferred to carry out extraction using an aqueous alcohol solution having a low to high concentration. Preferably, the concentration of the aqueous alcohol solution used in the step (B) is from 5 wt% to 30 wt%, and the concentration of the aqueous alcohol solution used in the step (C) is from 15 wt% to 50 wt%, and the concentration of the aqueous alcohol solution used in the step (D). The concentration of the aqueous alcohol solution used in the step (E) is from 50% by weight to 85% by weight, and the concentration of the aqueous alcohol solution used in the step (F) is from 70% by weight to 100% by weight. More preferably, the concentration of the aqueous alcohol solution used in the step (B) is from 5 wt% to 15 wt%, and the concentration of the aqueous alcohol solution used in the step (C) is from 25 wt% to 35 wt%, and the concentration of the aqueous alcohol solution used in the step (D) Is 45wt% to 55 wt%, the concentration of the aqueous alcohol solution used in the step (E) is from 65 wt% to 75 wt%, and the concentration of the aqueous alcohol solution used in the step (F) is from 90 wt% to 100 wt%.

Furthermore, in the citron extract of the present invention and the preparation method thereof, after the step (F), a step (G) may be further included: drying the citron extract.

In the camphor extract of the present invention and a method for preparing the same, each of the solid phase extract and the camphor extract is obtained by drying. Among them, the drying process may employ lyophilization, spray-drying, and evaporation. Preferably, a freeze-drying treatment is employed.

Further, the pharmaceutical composition of the present invention may further comprise: a pharmaceutically acceptable carrier. Here, the content of the camphor extract in the pharmaceutical composition of the present invention is preferably from 0.5 mg/ml to 5 mg/ml, more preferably from 0.5 mg/ml to 3 mg/ml. Further, the pharmaceutical composition or the extract of the camphor of the present invention is preferably used in an amount of 0.5 g to 10 g of the pharmaceutical composition or the extract of the camphor, per kg of the main system to be treated, and more preferably 0.5 g to 5 g of the medicine. Composition or citron extract.

Here, the carrier used in the pharmaceutical composition must be a "pharmaceutically acceptable carrier", that is, it must be compatible with the active principal component of the composition (preferably to stabilize the active principal component) and not cause damage to the subject. .

Fig. 1 is a graph showing the results of an oil droplet accumulation experiment of the present invention.

Fig. 2 is a graph showing the results of experiments on changes in body weight of mice of the present invention.

Fig. 3 is a graph showing the trend of weight gain of the mouse body weight change experiment of the present invention.

The first solid phase extract of the citron used in this example was purchased from Taiwan's citron Division (Kaohsiung, Taiwan), the preparation method is as follows.

The young leaves of the camphor were washed with water, and the water was mixed with the camphor leaves with 4 L of RO water per kg of camphor leaves, and boiled for 30 minutes to obtain a camphor extract. After that, the citron leaves were taken out, and the citron extract was concentrated by heating, and then filtered through a sieve (70 mesh) to a crude extract of citron leaves.

Then, the crude extract of the citronella leaves is reacted at a temperature of 121 ° C and a pressure of 1.2 kgw / cm ² for 20 minutes, and then freeze-dried to obtain a first solid phase extract.

The first solid phase extract was dissolved in the extract in a ratio of 10 ml of the extract per gram of the extract, and shaken at room temperature for 12 hours for extraction. After centrifugation, a supernatant and a precipitate are obtained. Here, the extract was a 10% aqueous solution of ethanol. After the precipitated portion is freeze-dried, a second solid phase extract can be obtained.

Then, the second solid phase extract is obtained by extracting the second solid phase extract in the same process as the extraction of the first solid phase extract. Here, the extract was a 30% aqueous solution of ethanol.

Then, the third solid phase extract is extracted by the same process as the extraction of the first solid phase extract to obtain a fourth solid phase extract. Here, the extract was a 50% aqueous solution of ethanol.

A fifth solid phase extract can be obtained by extracting the fourth solid phase extract in the same process as the extraction of the first solid phase extract. Here, the extract was a 70% aqueous solution of ethanol.

Finally, by extracting the fifth solid phase extract in the same process as the extraction of the first solid phase extract, a sixth solid phase extract, a so-called citron extract, can be obtained. Here, the extract was a 95% aqueous solution of ethanol.

20 μl of a 1 mg/ml aqueous solution of Toona sinensis extract was taken and injected into HPLC for division. HPLC analysis system is visible using Hitachi pump L7100 pump and Jasco UV1575 UV Light detector. The column is made of Ascentis 18, 15 cm x 4.6 mm I.D., 5 μm particles. The mobile phase was allowed to increase the methanol concentration from 20% to 100% in 20 minutes and the flow rate was 1 ml/min. Detection was performed at OD 280 nm to initially analyze the active ingredients. And known standards such as gallic acid, quercetin, methyl gallate, ethyl gallate, rutin Tea (atechin, epicatechin) peak comparison.

In addition, 20 μl of a 1 mg/ml aqueous solution of Toona sinensis extract was taken for LC-MS analysis. Here, the analysis was carried out using a 4000 Q Trap LC-MS tandem liquid chromatography mass spectrometer (Successful University Instrument Center), and the ion source was a nano-electrospray mass spectrometer (nano ESI) at a flow rate of 1 ml/min. And known standards such as gallic acid, quercetin, methyl gallate, ethyl gallate, rutin, The crest of catechin (epicatechin) is compared.

The results of the above HPLC and LC-MS analysis are shown in Table 1 below.

The total amount is the ratio of the components of the preliminary identification component of HPLC that does contain its components. For example, the ratio of gallic acid to HPLC is 21%, and the preliminary result is multiplied by the ratio of LC-MS identification of 41%, and the ratio is 8.5%.

As shown in the total amount of Table 1, the analyzed aqueous extract of camphor extract contained 8.5% gallic acid, 3.7% ethyl gallate, 2.6% methyl gallate, and 0.5%. Musk.

Therefore, after conversion, the solid citron extract comprises 57 parts by weight of gallic acid, 25 parts by weight of ethyl gallate, 20 parts by weight of methyl gallate, and 4 parts by weight of musk Prime.

Here, the oil droplet accumulation experiment was carried out using the precursor fat cell strain 3T3-L1. The 3T3-L1 cell line was cultured in DMEM medium containing 10% calf serum and 10 U/ml penicillin/streptin, cultured in a 37 ° C, 5% CO ₂ incubator, and the medium was changed every two to three days. . Subculture was performed when the cells reached 7 to 8 full. First, remove the culture solution and wash it with PBS buffer solution, add trypsin-EDTA in the incubator for 5 minutes. After the cells are completely detached, fully disperse the cells in DMEM medium, according to 1:4 The ratio of 1:6 (cell: culture solution) was further divided and cultured.

The 3T3-L1 cell line was cultured in DMEM medium containing 10% calf serum (BCS) to a confluence of 8 x 10 ⁴ cells, and then replaced with DMI differentiation induction medium (containing 10% fetal bovine serum DMEM). 1 μM corticosteroid (dexamethasone), 0.5 mM 3-isobutyl-methylxanthine, and 1.7 μM insulin (insulin).

After 48 hours of induction differentiation, the medium was replaced with the original medium, and the extract of citron (1.7 μmol/ml, 50 μg/ml, 100 μg/ml) containing 1.7 μmol/L of insulin, the differentiation-inducing agent DMI, or the 10 μM PPARγ expression promoter were added. PIO), replaced by 48 hours later 1.7 μmol/L insulin original medium. After 4 days of continuous culture, oil red (Oil red O) staining was performed. The procedure of oil red staining is as follows: the culture medium is washed with PBS buffer solution, and the cells are fixed with 10% formalin for 1 hour, washed with 60% isopropanol, and dried with oil red staining solution. (Oil red O working solution) was immersed for 10 minutes, and then washed with water, and the formation of red oil droplets was observed under a microscope. In addition, the moisture was removed, and after drying, it was dissolved in 100% isopropanol, and the absorbance at OD 500 nm was measured.

As a result, as shown in Fig. 1, the cell type of DMI and PIO was transformed from a spindle shape to a spherical shape, and the accumulation of oil droplets in the cells was clearly observed. Compared with the fat cells treated with DMI, the fat cells treated with the camphor extract did not have oil droplets.

The experiment of reducing the accumulation of oil droplets by the extract of the camphor of the present invention confirmed that the extract of the camphor can achieve lipolysis, thereby achieving the purpose of reducing body fat.

The experimental animals used in the present invention were 6-week-old male C57BL/6 mice, which were purchased from the Experimental Animal Center of the National Experimental Research Institute. The temperature of the animal room was maintained at 20±2°C, and the relative humidity was maintained at 50±20%. With the ventilation and air-conditioning equipment, the automatic timer was set to 7:00~19:00 as the illumination period, and the experimental animals were randomly divided into 6 groups. 10 in each group: After training for one week, divided into control group (n=10) and induction group (n=50), began to feed high-fat diet, the calorie source was 54% fat, 23% protein and carbohydrate.

After one week of experimental animal training, the body weight of the mice was determined, and the mice were weighed according to the body weight of the mice. The average body weight of each cage mouse was similar, and 5 cages were one cage. The feed, drinking water weight and animal body weight were measured weekly.

The results are shown in Figures 2 and 3, where CD indicates the normal diet group without high-fat diet, HFD indicates that the high-fat diet group was fed, and PIO indicates that PPARγ was fed. Ingredients and high fat feed groups. In addition, a further group is fed the toon extract and the high fat feed of the present invention.

As shown in Fig. 2, the weekly changes in body weight of each group showed a positive correlation, especially compared to the HFD group, when the 2 g/kg of the camphor extract of the present invention was fed at the eighth week, the mouse body weight The rate of increase has slowed significantly. Further, if it was observed that the body weight was subtracted from the body weight after the administration of the extract of the citron extract for eight weeks, it was found that the 2 g/kg of the citron extract of the present invention significantly slowed the weight gain as compared with the HFD group, as shown in Fig. 3.

From the mouse body weight change experiment, it was revealed that the toon extract of the present invention can achieve the same weight-reducing effect as PIO. Further, the toon extract of the present invention is a natural product and can be regarded as one of natural health foods.

The above-mentioned embodiments are merely examples for convenience of description, and the scope of the claims is intended to be limited to the above embodiments.

Claims (16)

A use of a camphor extract for preparing a medicament for reducing body fat, the camphor extract comprising: 40 to 75 parts by weight of gallic acid; 15 to 40 parts by weight of ethyl gallate; and 10 to 30 parts by weight of methyl gallate; wherein the extract of the camphor is obtained by the following steps: (A) providing a camphor leaf, and extracting the camphor leaf with water to obtain a camphor extract, and filtering the solution. The first extract of the camphor to obtain a first solid phase extract; (B) extracting the first solid phase extract with an aqueous alcohol solution to obtain a second solid phase extract; (C) extracting the second solid using an aqueous alcohol solution a phase extract to obtain a third solid phase extract; (D) extracting the third solid phase extract with an aqueous alcohol solution to obtain a fourth solid phase extract; (E) extracting the fourth solid using an aqueous alcohol solution a phase extract to obtain a fifth solid phase extract; and (F) extracting the fifth solid phase extract with an aqueous alcohol solution to obtain a citron extract; wherein the concentration of the aqueous alcohol solution used in the step (B) is The concentration of the aqueous alcohol solution used in the step (C) is 15 wt% to 30 wt%. % to 50% by weight, the concentration of the aqueous alcohol solution used in the step (D) is 30% by weight to 70% by weight, and the concentration of the aqueous alcohol solution used in the step (E) is 50% by weight to 85% by weight, and the step (F) is The concentration of the aqueous alcohol solution used is from 70% by weight to 100% by weight; 0.5 to 5 g of the camphor extract is administered per kg of the therapeutic subject to achieve a reduction in body fat by lipolysis. The use of the first aspect of the patent application, wherein the gallic acid content is 45 to 65 The content of the gallic acid ethyl ester is 20 to 30 parts by weight, and the methyl gallate content is 15 to 25 parts by weight. The use according to claim 2, wherein the gallic acid is 50 to 60 parts by weight, the gallic acid ethyl ester is 20 to 30 parts by weight, and the gallic acid is The content of the ester is from 15 to 25 parts by weight. The use of the third aspect of the patent application includes: 1 to 10 parts by weight of the ruthenium. The use of the first aspect of the invention, wherein the step (B), the step (C), the step (D), the step (E), and the aqueous solution of the alcohol used in the step (F) Each is independently selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, and mixtures thereof. The use of the first aspect of the invention, wherein the concentration of the aqueous alcohol solution used in the step (B) is 5 wt% to 15 wt%, and the concentration of the aqueous alcohol solution used in the step (C) is 25 wt% to 35 wt%. The concentration of the aqueous alcohol solution used in the step (D) is 45 wt% to 55 wt%, the concentration of the aqueous alcohol solution used in the step (E) is 65 wt% to 75 wt%, and the aqueous alcohol solution used in the step (F) The concentration is from 90% by weight to 100% by weight. The use of the first aspect of the invention, wherein the step (B), the step (C), the step (D), the step (E), and the aqueous solution of the alcohol used in the step (F) They are each independently an aqueous solution of ethanol. The use of claim 1, wherein after the step (F), a step (G) is further included: drying the citron extract. A use of a citron extract for the preparation of a medicament for reducing body weight, the citron extract comprising: 40 to 75 parts by weight of gallic acid; 15 to 40 parts by weight of ethyl gallate; and 10 to 30 parts by weight of methyl gallate; wherein the extract is obtained by the following steps: (A) providing a camphor leaf, and extracting the camphor leaf with water to obtain a first extract of the camphor, and filtering the first extract of the camphor to obtain a first solid phase extract; (B) extracting the first extract using an aqueous alcohol solution a solid phase extract to obtain a second solid phase extract; (C) extracting the second solid phase extract with an aqueous alcohol solution to obtain a third solid phase extract; (D) extracting the first extract using an aqueous alcohol solution a three solid phase extract to obtain a fourth solid phase extract; (E) extracting the fourth solid phase extract with an aqueous alcohol solution to obtain a fifth solid phase extract; and (F) extracting the aqueous solution with an alcohol a fifth solid phase extract to obtain a citron extract; wherein the concentration of the aqueous alcohol solution used in the step (B) is 5 wt% to 30 wt%, and the concentration of the aqueous alcohol solution used in the step (C) is 15 wt% to 50wt%, the concentration of the aqueous alcohol solution used in the step (D) is 30% by weight to 70% by weight, which is used in the step (E) The concentration of the aqueous solution is from 50% by weight to 85% by weight, and the concentration of the aqueous alcohol solution used in the step (F) is from 70% by weight to 100% by weight; from 0.5g to 5g of the extract of the medicinal herb per kg of the therapeutic body, the fat is decomposed The effect is to reduce body fat and achieve weight loss. The use according to claim 9, wherein the gallic acid content is 45 to 65 parts by weight, the gallic acid ethyl ester is 20 to 30 parts by weight, and the gallic acid A The content of the ester is from 15 to 25 parts by weight. The use according to claim 10, wherein the gallic acid content is 50 to The content of the gallic acid ethyl ester is 20 to 30 parts by weight, and the methyl gallate content is 15 to 25 parts by weight, based on 60 parts by weight. The use of the citron extract further comprises: 1 to 10 parts by weight of ruthenium, as claimed in claim 11 of the patent application. The use according to claim 9, wherein the step (B), the step (C), the step (D), the step (E), and the aqueous solution of the alcohol used in the step (F) are used. Each is independently selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, and mixtures thereof. The use according to claim 9, wherein the concentration of the aqueous alcohol solution used in the step (B) is 5 wt% to 15 wt%, and the concentration of the aqueous alcohol solution used in the step (C) is 25 wt% to 35 wt%. The concentration of the aqueous alcohol solution used in the step (D) is 45 wt% to 55 wt%, the concentration of the aqueous alcohol solution used in the step (E) is 65 wt% to 75 wt%, and the aqueous alcohol solution used in the step (F) The concentration is from 90% by weight to 100% by weight. The use according to claim 9, wherein the step (B), the step (C), the step (D), the step (E), and the aqueous solution of the alcohol used in the step (F) are used. They are each independently an aqueous solution of ethanol. The use of claim 9, wherein after the step (F), a step (G) is further included: drying the citron extract.